This invention relates to the first described pathogen-recognition molecule Nod1, which senses specifically Gram-negative bacteria through a peptidoglycan motif, muramyl tripeptide. More particularly, this invention relates to the modulation of Nod1 activity by a molecule related to the muramyl tripeptide (MTP). The invention also relates to a screening process for identifying a molecule capable of modulating Nod1 activity and the therapeutic use of such a molecule for modulating inflammation and/or apoptosis. The invention also relates to a new compound, which can be used for modulating inflammation and/or apoptosis or as an adjuvant agent.
In multicellular organisms, homeostasis is maintained by balancing the rate of cell proliferation against the rate of cell death. Cell proliferation is influenced by numerous growth factors and the expression of proto-oncogenes, which typically encourage progression through the cell cycle. In contrast, numerous events, including the expression of tumor suppressor genes, can lead to an arrest of cellular proliferation.
In differentiated cells, a particular type of cell death called apoptosis occurs when an internal suicide program is activated. This program can be initiated by a variety of external signals as well as signals that are generated within the cell in response to, for example, genetic damage. For many years, the magnitude of apoptotic cell death was not appreciated because the dying cells are quickly eliminated by phagocytes, without an inflammatory response.
The mechanisms that mediate apoptosis have been intensively studied. These mechanisms involve the activation of endogenous proteases, loss of mitochondrial function, and structural changes, such as disruption of the cytoskeleton, cell shrinkage, membrane blebbing, and nuclear condensation due to degradation of DNA. The various signals that trigger apoptosis are thought to bring about these events by converging on a common cell death pathway that is regulated by the expression of genes that are highly conserved from worms, such as C. elegans, to humans. In fact, invertebrate model systems have been invaluable tools in identifying and characterizing the genes that control apoptosis. Through the study of invertebrates and more evolved animals, numerous genes that are associated with cell death have been identified, but the way in which their products interact to execute the apoptotic program is poorly understood.
Caspases, a class of proteins central to the apoptotic program, are cysteine proteases having specificity for aspartate at the substrate cleavage site. These proteases are primarily responsible for the degradation of cellular proteins that lead to the morphological changes seen in cells undergoing apoptosis. For example, one of the caspases identified in humans was previously known as the interleukin-1β (IL-1β) converting enzyme (ICE), a cysteine protease responsible for the processing of pro-IL-1β to the active cytokine.
Many caspases and proteins that interact with caspases possess domains of about 60 amino acids called a Caspase Recruitment Domain (CARD). Others have postulated that certain apoptotic proteins bind to each other via their CARDs and that different subtypes of CARDs may confer binding specificity, regulating the activity of various caspases, for example.
Innate immunity to bacterial pathogens relies on the specific sensing of pathogen-associated molecular patterns (PAMPs) by pattern-recognition molecules (PRMs). In mammals, Toll-like receptors (TLRs) represent the most extensively studied class of PRMs, which have been shown to sense various PAMPs, such as lipopolysaccharide (LPS), peptidoglycan (PGN), lipoproteins, double-stranded RNA and CpG DNA (S. Akira et al., Nat. Immunol., 2, 675, 2001; R. Medzhitov, Nat. Rev. Immunol., 1, 135, 2001). While TLRs are mainly expressed at the plasma membrane, it has been recently proposed that the Nod molecules, a family of intracellular proteins including Nod1/CARD4 and Nod2/CARD15, could represent a new group of PRMs that sense bacterial products within the cytoplasmic compartment, thus allowing to detect the presence of intracellular invasive bacteria (N. Inohara et al., J. Biol. Chem. 274, 14560, 1999; J. Bertin et al., J. Biol. Chem., 274, 12955, 1999; N. Inohara et al., J. Biol. Chem., 276, 2551, 2001; S. E. Girardin et al., EMBO Rep., 2, 736, 2001; Y. Ogura et al., J. Biol. Chem., 276, 4812, 2001; Y. Ogura et al., Nature, 411, 603, 2001; J. P. Hugot et al., Nature, 411, 599, 2001).
The partial sequences (cDNA and protein) of Nod1, also named CARD4 for Caspase Recruitment Domain, have been disclosed in patent application Ser. No. 09/019,942, filed Feb. 6, 1998, and now granted as U.S. Pat. No. 6,033,855. Furthermore, Bertin et al. (1999) disclosed the entire amino acid sequence of CARD4 and one of its functions, already mentioned in the above-patent: CARD4 coordinates NF-κB and apoptotic signaling pathway. Girardin et al. (2001) disclosed that CARD4/Nod1 mediates NF-κB activation by invasive Shigella flexneri. In this article, the interaction between S. flexneri LPS and CARD4 is especially studied.
Stimulation of Nod1/CARD-4 activity is desirable in situations in which CARD-4 is abnormally downregulated and/or in which increased CARD-4 activity is likely to have a beneficial effect. Conversely, inhibition of CARD-4 activity is desirable in situations in which CARD-4 is abnormally upregulated, e.g., in myocardial infarction, and/or in which decreased CARD-4 activity is likely to have a beneficial effect. Since CARD-4 may be involved in the processing of cytokines, inhibiting the activity or expression of CARD-4 may be beneficial in patients that have aberrant inflammation.
It has recently been shown that Nod1 senses the presence of the Gram-negative pathogen, Shigella flexneri, within the cytoplasmic compartment of epithelial cells (S. E. Girardin et al., 2001), and it was hypothesized that the detected PAMP was LPS since commercial preparations of LPS were shown to activate Nod1 (N. Inohara et al., J. Biol. Chem., 276, 2551, 2001). However, as these LPS often contain bacterial cell wall contaminants, there is a need in the art for more detail on whether a particular molecular motif or motifs are actually detected by Nod1 and modulate Nod1 activity.